The interaction between abiotic factors and population genetic
processes is important in determining the course of evolutionary change.
Temperature fluctuations in particular have been emphasized as a stress
factor that can affect the rate and direction of evolutionary change
(Parsons 1987; Hoffmann and Parsons 1991; Lenski and Bennett 1993). It
has been proposed that selection conditions that border on the limits of
an organism's ability to persist may result in rapid evolutionary
responses (Hoffmann and Parsons 1991; Bennett et al. 1992; Howarth
1993). In addition, if there is genetic coupling between characteristics
that govern performance at the high and low ends of the range for an
environmental factor, then adaptation to one extreme may be associated
with trade-offs that reduce performance in other environments (Huey and
Kingsolver 1989) and could channel the future evolution of the niche in
one direction or another (Brooks and McLennan 1991). We have designed an
experimental system that allows us to monitor evolutionary adaptation in
the bacterium Escherichia coli in response to changes in an important
environmental factor, temperature, and we evaluate the results in light
of such evolutionary predictions.

This particular experiment arose from previous studies that employed
natural selection in the laboratory to examine adaptation of E. coli
populations to different thermal regimes (Bennett et al. 1990, 1992;
Bennett and Lenski 1993). In those studies, a single bacterial clone,
with an evolutionary history at 37 [degrees] C, was used to found
replicate experimental lines that were propagated in four thermal
regimes: 32, 37, and 42 [degrees] C, and a daily alternation between 32
and 42 [degrees] C. After 2000 generations, these experimental groups
were found to have adapted (as indicated by improved fitness relative to
their common ancestor) to their particular selective thermal
environments. The groups were not found, however, to have significantly
altered their ancestral niche (defined as the range of temperatures over
which they could maintain their populations during daily serial dilution
culture). The lower boundary of this common thermal niche was found to
be approximately 19.5 [degrees] C. We therefore undertook this
experiment in which a new experimental group was founded from the
original common ancestor and propagated at 20 [degrees] C for 2000
generations. This experimental group thus has a parallel evolutionary
history to our original four experimental groups and supplements our
original set to span the entire ancestral thermal niche. Its creation
permits us to address a series of questions about the rate and extent of
adaptation at the lower thermal niche edge and its correlated responses.
In addition to the creation of the 20 degrees C group, the replicate
lines of the four original experimental groups were moved to the 20
[degrees] C environment and propagated for a further 2000 generations in
this novel thermal environment. These groups are designated 32/20,
37/20, 42/20, and 32-42/20 [degrees] C to indicate their historical
thermal exposure. The thermal histories of each of these lines has thus
been controlled for 6000 generations; for example, the 32/20 [degrees] C
group had a history of 2,000 generations at 37 [degrees] C, followed by
2000 generations at 32 [degrees] C, and now a further 2000 generations
at 20 [degrees] C. A phylogeny, thermal history, and taxonomy of these
experimental lineages is presented in Figure 1.

Analysis of these groups permits us to address several questions that
are relevant to adaptive evolution in general and evolutionary responses
to temperature in particular. First, do populations adapt more rapidly
to temperatures at the extreme limits of their thermal niche? In our
previous studies (Bennett et al. 1990, 1992), we observed that the 42
[degrees] C group adapted much more rapidly and achieved a much greater
eventual increase in fitness than the groups maintained at lower, and
more moderate, temperatures. We proposed that one explanation for this
differential response to natural selection could be the severity of
selection at the limits of thermal tolerance. Alternatively, it might
reflect some asymmetry between higher and lower temperatures in the
stringency of selection. The upper thermal boundary is considerably
sharper than the lower, where population growth gradually slows at
increasingly colder temperatures until the population can no longer
persist. If proximity to the edge of a niche boundary per se results in
more rapid evolution, we would expect to see more rapid and pronounced
fitness responses to both niche extremes. In this case, adaptation to 20
[degrees] C should be similar in rapidity and extent to that observed at
42 [degrees] C, both being greater than adaptation to an intermediate
temperature such as 32 [degrees] C. In addition, this project was
intended to explore further the existence of correlated tradeoffs
associated with thermal adaptation. Does adaptation to an environmental
variable impact the breadth and shape of an organism's niche with
respect to that variable? Finally, the design of this experiment allowed
us to examine whether evolutionary history had an effect on the rate
and/or ultimate extent of subsequent adaptation to a novel environment.
Does, for instance, prior adaptation to a moderately low temperature (32
[degrees] C) accelerate adaptation at 20 [degrees] C relative to prior
adaptation to a high temperature (42 [degrees] C)?

MATERIALS AND METHODS

Bacterial Strains and Media. - The ancestral strain used in this
study was a clone isolated from a population of E. coli B that had been
maintained by serial propagation in glucose-limited medium for 2000
generations at 37 [degrees] C (Lenski et al. 1991). An isogenic
derivative of that clone that can utilize the sugar L(+)arabinose
([Ara.sup.+]) was obtained by spontaneous mutation. The [Ara.sup.+] and
[Ara.sup.-] phenotypes can be distinguished by plating on tetrazolium
arabinose (TA) indicator agar. Six replicate lines were initiated from
these clones, three of each marker state. The marker allowed us to
monitor the cultures for evidence of cross-contamination between
populations as well as to compete the evolving lines in mixed culture
with the ancestor bearing the opposite marker. The marker itself has
been shown to be effectively neutral under the conditions employed in
this study. These six lines are designated as the 20 [degrees] C group
[ILLUSTRATION FOR FIGURE 1 OMITTED].

An additional four groups of six populations each were initiated with
clonal isolates obtained by Bennett et al. (1992). In that previous
experiment, the 24 lines underwent 2000 generations of evolution
parallel to that described above but at different temperatures. Clonal
isolates from the populations at the end of that study were used to
found new populations that were moved to the 20 [degrees] C incubator
and propagated in parallel with the six lines described above. These
four groups of six lines are designated as the 32/20, 37/20, 42/20, and
32-42/20 [degrees] C groups to indicate the temperature at which they
were previously exposed (32, 37, 42, and daily alternation between 32
and 42 [degrees] C, respectively) and their subsequent exposure to 20
[degrees] C [ILLUSTRATION FOR FIGURE 1 OMITTED].

The culture media used in this study was Davis minimal (DM)
supplemented with glucose (25 [[micro]gram]/ml) (Lenski et al. 1991).
The common ancestor as well as all clonal isolates obtained in this
study are stored for future analysis in a 12% glycerol suspension at -80
[degrees] C.

Culture Conditions. - All cultures described here were initiated from
single clones and propagated by daily transfer of 0.1 ml of each culture
into 9.9 ml of fresh liquid media. The cultures were incubated at 20
[degrees] C unless otherwise stated and shaken at 120 rpm. The temporal
and spatial variation in the incubator was within [+ or -] 0.5 [degrees]
C. Bacterial populations were enumerated by spreading diluted cultures
on TA indicator agar plates, which were incubated at 37 [degrees] C for
one day. The plating medium and temperature represents an arbitrary
environment in which to enumerate the abundance of a population.

The evolving lines in all five experimental groups were plated on TA
agar after 100, 200, 300, 600, and every succeeding 200 generations
through a total of 2000 generations. For each line, a single colony was
chosen at random. These clonal isolates were regrown in medium
containing glycerol and then stored at -80 [degrees] C. After 300
generations, several of the populations began to show reduced plating
efficiency at 37 [degrees] C. To reduce the possibility of selecting
clones from only a subset of the population, clones were isolated from
plates incubated for four days at 20 [degrees] C from that point onward.

Measurements of Relative Fitness. - Relative fitness was measured by
direct competition between each evolved line and the ancestral clone
with the opposite Ara marker state. Cultures of the populations to be
competed were inoculated from freezer stocks into a rich broth (LB)
(Miller 1972) and grown overnight at 37 [degrees] C. These cultures were
then diluted and grown in DM for two days at the appropriate temperature
to allow the cultures to become comparably acclimated to the environment
(e.g., Leroi et al. 1994). After this preconditioning, the 24-h cultures
of each pair of competitors were mixed 1:1 and diluted 100-fold into
fresh media. Samples of this mixture were taken immediately and again
after 24 h of growth, diluted and plated on TA agar to estimate the
initial and final densities of each competitor. Relative fitness (W) is
defined as the ratio of the number of doublings achieved during the
competition by the experimental line being tested and its ancestor
(Lenski et al. 1991). This fitness was calculated as follows:

W = [log.sub.2]([N.sub.t]/[N.sub.0])/[log.sub.2]([A.sub.t]/[A.sub.0])

where N and A are the densities of the evolved and ancestral lines,
respectively, and the subscripts 0 and t indicate the initial and final
time points (i.e., 0- and 24-h samples). A difference in plating
efficiency between the competitors will not affect our estimates of
relative fitness as long as plating efficiency is the same at each time
point.

Measurements of Absolute Fitness. - Absolute fitness was estimated by
regressing the natural logarithm of population density immediately prior
to serial dilution against time for each of the ancestral and evolved
genotypes in pure culture in the same medium in which they had evolved
(see above) but at a variety of different temperatures (see Bennett and
Lenski 1993). The slope of the regression is equivalent to the
genotype's Malthusian parameter. In order to maintain a constant
population size under these conditions, the population must grow at
least fast enough to compensate for the 100-fold daily dilution. As long
as the population is at its maximum density for the given resource level
at the beginning of the experiment, the Malthusian parameter cannot be
significantly greater than zero. A slope less than zero indicates that
the population is growing more slowly than the dilution rate imposed by
the environment. A slope of -4.6 (In 0.01) indicates that the population
is neither growing nor dying, but is simply being diluted from the
culture. These experiments were carried out in a shaking water bath
incubator with temporal fluctuations of [+ or -]0.1[degrees] C. The
cultures were removed from the incubator for only a few minutes to take
a sample of each and transfer it to fresh media.

RESULTS

Rate and Extent of Adaptation of the 20 [degrees] C Group. - The
fitness responses observed during 2000 generations of evolution at 20
[degrees] C are shown in Figure 2 for the six lines of the 20 [degrees]
C group. Mean relative fitness increased during the course of the
experiment to a final value at 2000 generations of 1.08 ([+ or -]0.014
SE). An analysis of variance failed to demonstrate any significant
heterogeneity among lines in the magnitude of their relative fitness at
the end of the experiment ([F.sub.5,30] = 1.338; P = 0.275). The mean
relative fitness for each line was regressed over time using a linear
model to estimate the average rate of change in fitness over the entire
2000 generations; the intercept was constrained to a value of 1.0, which
is by definition the ancestral fitness. The average rate of change in
relative fitness for the group was 4.658 ([+ or -]0.104 SE) X
[10.sup.-5] per generation.

1 In every case, the mean difference is calculated by subtracting
the value for the second group in the comparison from the value for
the 20 [degrees] C group.

2 Two-tailed probabilities were calculated for each comparison (I)
with six lines per group ([n.sub.1] = [n.sub.2] = 6) and by
applying
the sequential Bonferroni technique to each set of four comparisons
(SB). In the latter case, an (*) indicates that the null hypothesis
of no difference is rejected at P [less than] 0.05 and (**) at P
[less than] 0.01.

3 Using data from assays performed by A. F. Bennett and R. E.
Lenski
(1996).

4 Using data from Bennett et al. (1992).

One of the primary motivations for this study was to determine
whether the fitness response to natural selection at the lower limit of
this bacterium's thermal niche would be similar to that observed in
response to natural selection at the upper thermal limit (Bennett et al.
1992). To test this hypothesis, we compared both the rate of adaptation
and the final mean relative fitness of the 20 [degrees] C group with
published estimates for groups derived from the same ancestral strain in
the same environment but at higher temperatures, including the ancestral
temperature (37 [degrees] C) and the upper thermal limit for this
bacterium (42 [degrees] C) (Bennett et al. 1992; Bennett and Lenski
1996). The results are summarized in Table 1. The rates of adaptation
and the final fitness for the six replicate lines of the 20 [degrees] C
group were compared with the values for each of the parallel groups by
Mann-Whitney tests with probability levels corrected using the
sequential Bonferroni technique (Rice 1989). The increase in the final
fitness of the 20 [degrees] C group, representing adaptation to a novel
environment, was significantly higher than was observed for the 37
[degrees] C group, which was maintained in the ancestral environment.
The fitness of the 20 [degrees] C group was, however, significantly
lower than the 42 [degrees] C and 32-42 [degrees] C groups even after
applying the sequential Bonferroni correction to the probability level.
There was no significant difference between the fitness response at 20
[degrees] C and that at the more moderate (but also novel) temperature
of 32 [degrees] C. With respect to the rates of adaptation, only the
comparison with the 42 [degrees] C group was significant. However, this
comparison is the one of primary interest with respect to the
hypothesized effect of the niche edge. Neither the rate nor final extent
of adaptation supports the hypothesis of a response to selection at the
lower thermal niche boundary comparable in magnitude to that at the
upper niche boundary. Rather, there appears to be an asymmetrical
response to selection at the upper and lower boundaries, with the high
rate of change in fitness that was previously observed (Bennett et al.
1992) being unique to the upper thermal boundary.

Thermal Niche Modification. - The absolute fitnesses of the six lines
of the 20 [degrees] C group and their ancestor are plotted as a function
of temperature in Figure 3. The absolute fitness of the ancestor was
compared with the estimates for the lines of the 20 [degrees] C group by
a Mann-Whitney test at assay temperatures near the boundaries of the
ancestral thermal niche. All six lines are able to maintain their
population at a temperature of 18 [degrees] C, 1.5 [degrees] C below the
ancestral limit. The difference between the 20 [degrees] C group (based
on six replicate lines) and their ancestor (based on the two replicate
ancestors of opposite marker types) was significant (P = 0.046) at that
temperature. At 16.8 [degrees] C, neither the 20 [degrees] C group nor
the ancestor is able to maintain their population density in this serial
dilution regime, but the former has a greater absolute fitness than does
the ancestor (P = 0.046, with a mean difference of 1.14 [day.sup.-1]).
At the upper niche boundary, there is considerably more variation in
fitness among the six replicate 20 [degrees] C-selected lines. At 41.5
[degrees] C, the absolute fitness of the 20 [degrees] C group as a whole
does not differ from the ancestor (P = 0.096); however, four of the six
lines are unable to maintain their population density at that
temperature. At 42 [degrees] C, the ancestor is able to sustain
sufficient growth to replace its population every 24 h, but all six of
the experimental lines are unable to persist; this difference is
significant (P = 0.046) by the Mann-Whitney test. In the case of at
least two of the six lines, the rate of extinction was even greater than
the dilution rate during serial transfer, indicating that these lines
were not only unable to grow but also must have experienced some cell
death at that temperature.

Correlated Fitness Responses. - The mean relative fitness of the 20
[degrees] C group was measured after 2000 generations at temperatures
spanning the original thermal niche of the ancestor (Fig. 4).
Correlation coefficients between mean relative fitness and assay
temperature were calculated for each of the six lines. Three of the six
correlations were significant (P [less than] 0.05; five temperatures and
n - 2 = 3 df). This result suggests that most of the adaptation was
specific to growth at lower temperatures rather than to the general
culture conditions. in fact, adaptation of the 20 [degrees] C group
resulted in a significant decrease in fitness at 40 [degrees] C (W =
0.83 [+ or -] 0.05 SE; two-tailed P = 0.024).

Effect of Thermal History. - The fitness responses observed over the
course of 2000 generations for the four groups with histories of
evolution at different temperatures (32/20, 37/ 20, 42/20, and 32-42/20
[degrees] C groups) are shown in Figures 5A-D. A linear model was
employed, as described above for the 20 [degrees] C group, to estimate
the rate of change in fitness for each line during this time period.
Because these lines were derived from the common ancestor 2000
generations before the start of this experiment, we could not assume
that their initial fitnesses at 20 [degrees] C were equal to the
ancestor. Therefore, relative fitnesses at 20 [degrees] C at time 0 were
not constrained to 1.0. The means and standard errors of these rates are
summarized in Table 2. The standard errors are calculated based on the
six replicate lines within each group. All four groups underwent
statistically significant gains in relative fitness at 20 [degrees] C.
An analysis of variance failed to detect any heterogeneity in the rate
of change in fitness among the four groups ([F.sub.3,20] = 0.423; P =
0.74). Inclusion of the 20 [degrees] C group in this analysis likewise
fails to detect significant differences among rates of adaptation
([F.sub.4,25] = 2.381; P = 0.08).

DISCUSSION

An organism's potential niche is determined by the range of
different environmental factors (e.g., temperature, pH, and salinity) in
which a population can grow and persist. Suboptimal conditions in
environments at the periphery of an organism's normal range or
during episodes of environmental change are often physiologically
stressful. Parsons (1987) suggested that abiotic, particularly climatic,
stresses magnify phenotypic variation and can result in elevated rates
of evolutionary change. The ability to maintain replicated populations
of bacteria in well-controlled environments for evolutionarily relevant
lengths of time has allowed us to make detailed observations of this
phenomenon. In addition, we have been able to investigate the nature of
genetic correlations between performance in different environments. That
is, we can ascertain whether adaptation to a marginal environment will
influence an organism's performance within its ancestral range as
well as its potential for future adaptation to a changing environment.

Ambient temperature is an important environmental factor even for
enterobacteria, which must be capable of transmission to new hosts and
therefore persist for extended periods of time outside their host
environment. In addition, the enteric bacteria are resident in many host
species with a relatively wide range of body temperatures. Bennett et
al. (1992) employed natural selection in the laboratory to study
adaptation of initially identical populations of E. coli to several
environments that differed only in temperature. They observed a
significantly more rapid response to natural selection at a temperature
very near the upper limit of thermal tolerance (42 [degrees] C) when
compared with the response to selection at a moderate temperature (32
[degrees] C), even though both temperatures were equidistant from the
ancestral temperature (37 [degrees] C). Despite an average increase in
fitness of almost 50%, the group of lines evolved at 42 [degrees] C
showed no significant loss of fitness at much lower temperatures
(Bennett and Lenski 1993).

[TABULAR DATA FOR TABLE 2 OMITTED]

The current study, designed to test the generality of the previous
results by repeating the experiment near the lower thermal niche
boundary (20 [degrees] C), produced quantitatively and qualitatively
very different results. These may be summarized in three major
conclusions. First, occupation of a habitat in which the environment is
marginal for persistence of a population is not a sufficient condition
to explain an elevated rate of adaptation. During 2000 generations of
evolution at 20 [degrees] C, the bacterium in this study adapted to its
environment with an average increase in fitness relative to the common
ancestor of only 8%. The average rate of increase in fitness at this
temperature was not significantly different from that observed at a
novel temperature much closer to the middle of the ancestral thermal
niche (32 [degrees] C) and was significantly lower than that observed
near the upper thermal limit (42 [degrees] C). Our results thus indicate
that neither simple distance from the ancestral environment nor
proximity to the edge of the niche are sufficient indicators of
exceptionally high rates of evolutionary change.

The second conclusion is that performances at higher and lower
temperatures in the 20 [degrees] C lines are genetically correlated.
Associated with the increase in relative fitness at 20 [degrees] C was a
decrease in fitness at higher temperatures (by 17% when assayed at 40
[degrees] C). In addition to the relative fitness response, both the
upper and lower limits of thermal tolerance, as measured by the ability
of a population to persist in the serial transfer environment, shifted
downward by 1-2 [degrees] C. These observations of trade-offs associated
with adaptation to 20 [degrees] C are again in contrast to the lack of
any general observation of trade-offs associated with adaptation to
higher temperatures (Bennett et al. 1992; Bennett and Lenski 1993). A
related study in Drosophila melanogaster (Huey et al. 1991) demonstrated
a correlated effect of natural selection at intermediate temperatures on
tolerance to heat shock, however, they observed a significant increase
in tolerance to high temperatures rather than a trade-off. Many
evolutionary ecology models (e.g., Lynch and Gabriel 1987) assume that
tradeoffs will be associated with adaptation. Our results illustrate
that any assumptions about trade-offs may depend not only on the
particular environmental factor that is acting as the selective agent
but even on the direction of change in that environmental factor.

We have defined the thermal niche of a population as the range of
temperatures over which it can sustain itself indefinitely, given a
particular set of other environmental conditions including resource
level and density-independent mortality (due to serial dilution).
Evidently, the evolutionary responses to the lower and upper edges of
the thermal niche are quite different, both in terms of its rapidity and
the effects of that evolution on the thermal niche itself. Why were
there these differences? We can only speculate, but it may be important
that the upper boundary is extremely sharp and characterized by a sudden
shift from rapid growth to marked death between 42 and 44 [degrees] C
(see [ILLUSTRATION FOR FIGURE 4 OMITTED] in Bennett and Lenski 1993). By
contrast, the lower niche boundary is characterized by a gradual
reduction in growth rate, which at some point simply becomes
insufficient to offset the losses due to serial dilution. That is, in
contrast to temperatures just beyond the upper edge, there does not
appear to be any sudden dramatic change in cell physiology just below
the lower edge.

A third conclusion is that, despite our demonstration of genetic
correlations between performance at different temperatures, there was no
discernible effect of selection history on adaptation to this novel
thermal environment. Other studies have shown that responses to uniform
artificial selection can amplify historical differences in the value of
some traits between samples from natural populations. For example,
populations of D. melanogaster and Drosophila pseudoobscura derived from
different localities exhibited heterogeneous responses to artificial
selection for ethanol knockdown resistance and correlated traits (Cohan
and Hoffmann 1986; Hoffmann and Cohan 1987). While 2000 generations may
not have been long enough for strong historical contingencies to develop
in our bacterial lines, we have demonstrated elsewhere that a trait that
is not significantly correlated with Darwinian fitness was more
influenced by historical contingencies than was fitness itself
(Travisano et al. 1995).

Asymmetries in Evolutionary Responses. - The different responses to
natural selection at the upper and lower thermal niche boundaries of the
E. coli populations described here provide a new example of an
asymmetrical evolutionary response to selection in alternate
environments. Other examples include an experimental evolution project
involving adaptation of bacterial populations to alternate resources
(Travisano 1993). In that case, adaptation to growth in a
glucose-limited environment resulted in significant variation in fitness
in a maltose-limited environment. Adaptation to growth in the
maltose-limited environment, on the other hand, resulted in no increased
variation in fitness in glucose. A third example is derived from data on
parasitic wasps, Aphytes, artificially selected for heat and cold
tolerance (White et al. 1970; Huey and Kingsolver 1989). Selection for
cold tolerance increased both cold and heat tolerance, whereas selection
for heat tolerance increased only heat tolerance with no effect on cold
tolerance. In addition to laboratory studies, a comparison of
prokaryotes isolated from high and low temperature environments suggests
that such asymmetries occur in response to selection pressures in
natural environments as well. Many of the prokaryotes isolated from high
temperature environments are obligate thermophiles, whereas those
resident in low temperature environments are not obligate psychrophiles
and generally grow optimally at much higher temperatures (Knoll and
Bauld 1989). Whether these differences reflect past selective pressures
during the course of Earth's history or underlying asymmetry in the
connections between metabolic processes that are important at high and
low temperatures is only speculation at this point. If the latter is
true, are these asymmetries the result of inherent physical or
biochemical constraints or has past selection favored an asymmetric
design of the metabolic architecture? A corollary to this study might be
to look at correlated effects of temperature-sensitive mutations on
fitness at other temperatures.

Charting the path between the phenotypic adaptations responsible for
fitness differences and the underlying biochemical and genetic changes
that have occurred is likely to be difficult. Dykhuizen and Dean (1990),
however, demonstrated that a simple model mapping enzyme kinetic
parameters to fitness for the lactose operon could account for seemingly
complex phenomena such as genotype-by-environment interactions. Using
their model, one can see how those genotype-by-environment interactions
can in turn influence the order in which particular metabolic steps are
targeted by natural selection. Thus, the asymmetrical nature of our
observations on thermal adaptation may hint at further information
regarding the underlying metabolic architecture that is the source of
the phenotypic differences.

Finally, an analysis of the phenotype of experimentally evolved lines
in different environments has the potential to lead to identification of
the molecular basis of the adaptations. Further analyses of these lines
promises to expand our understanding of natural selection to include not
just the results of selection but also the nature of the genetic
variation and its relationship to the phenotypic variation upon which
selection operates.

ACKNOWLEDGMENTS

This research was supported by Research Excellence Funds from the
State of Michigan, the NSF Center for Microbial Ecology (BIR-9120006),
and an NSF Grant (IBN-9208662) to A.F.B. and R.E.L. We thank L. Ekunwe
for assistance in the laboratory.